Identification and validation of reference genes for real-time RT-PCR in Aphelenchoides besseyi.

Fragments of four candidate reference genes of Aphelenchoides besseyi, including actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin conjugating-3 enzyme (UBC) and alpha-tubulin (α-tubulin) were cloned from the transcriptome database of A. besseyi. The expression level of these four candidate reference genes and a commonly used reference gene of A. besseyi (18S rRNA) in three experimental conditions, including the four life stages (female, male, juvenile and egg) of two populations and the mixed-stage nematodes of four populations with different origins and hosts were analyzed by RT-qPCR. The expression stability of the five candidate reference genes under the three experimental conditions was analyzed by ΔCt, geNorm, NormFinder and RefFinder respectively. The analysis results of ΔCt, geNorm, NormFinder and RefFinder all indicated that UBC was the gene with the highest average ranking of stability. In conclusion, the expression stability of UBC was optimal under the three experimental conditions, indicating that UBC could be used as a suitable reference gene instead of 18S rRNA in the RT-qPCR analysis for A. besseyi.